Regulation of Evi-1 function via the corepressor CtBP

描述（由申请人提供）：转录辅阻遏物CtBP参与发育、细胞周期调控和转化的重要途径。 其中一个这样的途径涉及癌蛋白Evi-1，它与人类急性骨髓性白血病有关。 最近，主要研究者证明，CtBP是由烟酰胺腺嘌呤二核苷酸，NAD和NADH的方式，调节其与各种转录抑制因子相互作用的能力。 这项工作提出了一种全新的转录调控机制，其中阻遏物：辅阻遏物复合物的形成受游离NAD/NADH核水平变化的调节，CtBP作为转录的氧化还原传感器。 目前的项目的主旨是通过测量细胞核中游离烟酰胺腺嘌呤二核苷酸的水平，建立这种调节机制对人类转化蛋白Evi-1的性质的影响，并阐明CtBP介导的转录抑制机制，来扩展该模型。 提出了三个具体目标：1）确定烟酰胺腺嘌呤二核苷酸的游离核浓度是否适合其在CtBP调节中的拟议作用。 NAD和NADH对CtBP的亲和力将在体外测量，并与它们的游离核浓度进行比较，所述游离核浓度使用双光子荧光显微术在体内测定。2）确定CtBP感测核氧化还原状态的能力是否调节人癌蛋白Evi-1的功能，并测试CtBP中NAD/NADH结合位点的阻断剂是否抑制Evi-1功能，3）鉴定人细胞CtBP复合物的组分并确定这些组分对转录抑制的贡献。 通过NAD/NADH诱导的CtBP变构效应调节Evi-1功能可能会影响细胞转化过程。 因此，阻断NAD/NADH诱导的变化可能对某些类型的白血病有治疗作用。 通过指导的职业发展计划，主要研究者将获得基因调控领域的熟练程度，因为它涉及到分子肿瘤学。 导师的实验室和机构拥有丰富的智力和物质资源，为促进主要研究者向独立科学家过渡提供了良好的环境。

DESCRIPTION (provided by applicant): The transcriptional corepressor CtBP participates in pathways important for development, cell cycle regulation, and transformation. One such pathway involves the oncoprotein Evi-1, which has been linked to human acute myelogenous leukemia. Recently, the principal investigator demonstrated that CtBP is regulated by the nicotinamide adenine dinucleotides, NAD and NADH, in a manner that regulates its ability to interact with a variety of transcriptional repressors. This work suggested a completely novel mechanism for transcriptional regulation, in which the formation of a repressor:corepressor complex is regulated by changes in the nuclear levels of free NAD/NADH, with CtBP serving as a redox sensor for transcription. The main thrust of the current project is to extend this model by measuring the levels of free nicotinamide adenine dinucleotides in the nucleus, establishing the impact of this regulatory mechanism on the properties of the human transforming protein Evi-1, and elucidating the mechanism of CtBP-mediated transcriptional repression. Three specific aims are proposed: 1) Determine whether the free nuclear concentrations of nicotinamide adenine dinucleotides are appropriate for their proposed role in CtBP regulation. The affinities of NAD and NADH for CtBP will be measured in vitro and compared to their free nuclear concentrations, determined in vivo using two-photon fluorescence microscopy, 2) Establish whether the ability of CtBP to sense the nuclear redox state regulates the function of human oncoprotein Evi-1 and test whether blockers of the NAD/NADH binding site in CtBP inhibit Evi-1 function, 3) Identify components of the human cellular CtBP complex and determine the contributions of these components to transcriptional repression. Regulation of Evi-1 function through the proposed NAD/NADH-induced allosteric effect on CtBP is likely to affect the course of cell transformation. Blockers of the NAD/NADH-induced change may therefore be therapeutically useful in certain types of leukemia. Through a mentored career development plan, the principal investigator will gain proficiency in the field of gene regulation as it relates to molecular oncology. The mentor's laboratory and institution are rich in intellectual and physical resources that provide an excellent environment for facilitating the principal investigator's transition to independent scientist.